Abstract: (no more than 30 lines) Aberrant ubiquitination processes are tightly associated with various human diseases including cancer. The Anaphase Promoting Complex (APC) has been documented to regulate cell cycle progression through binding activator proteins Cdh1 and Cdc20 at different cell cycle stages to promote APC-dependent ubiquitination and destruction of their substrates. Recent studies revealed that APCCdc20 is an attractive anti-cancer target and depletion of Cdc20 suppresses in vivo tumorigenesis in part by triggering mitotic arrest and subsequent apoptosis. We have recently reported the pro-apoptotic protein Bim as a novel APCCdc20 substrate, which supports the notion that Cdc20 is an oncogene. However, how Cdc20 is controlled by upstream regulator(s) and how the Cdc20-Bim signaling axis contributes to chemoradio-resistance in vivo remains largely unaddressed. Our preliminary results showed that the CUL3SPOP E3 ligase specifically targets Cdc20 for ubiquitination and subsequent destruction to attenuate its oncogenic activity. Furthermore, we demonstrated that APCCdc20 governs the ubiquitination and subsequent destruction of Bim in a D-box-dependent manner. Notably, human Adult-T-cell-Leukemia cells that acquire elevated APCCdc20 activity via expressing the Tax-viral- oncoprotein, exhibit reduced Bim levels and corresponding resistance to anti-cancer reagents. Conversely, depletion of Cdc20 in various human cancer cell lines sensitized their response to various chemotherapeutic drugs. More importantly, Cdc20 and multiple APC-core components were identified in an siRNA screen that upon knockdown sensitizes head-and-neck-cancer cells to ?-irradiation in a Bim-dependent manner. Based on our preliminary data, we hypothesize that SPOP is a novel upstream negative regulator of Cdc20 stability and oncogenic functions, and APCCdc20 negatively governs the induction of apoptotic pathway in response to anti- mitotic agents and ?-irradiation in part through targeting Bim for ubiquitination and destruction. We will examine our hypothesis by accomplishing the following three specific aims. In Aim #1, we will determine the molecular mechanisms underlying SPOP-mediated ubiqutination and degradation of Cdc20 and to examine whether SPOP deficiency in head and neck cancer leads to elevated tumorigenesis in part by Cdc20 accumulation. Our proposed work in Aim #2 will determine the physiological role of APCCdc20 in governing cellular apoptosis in vivo by regulating the abundance of Bim and other Cdc20 substrates. These proposed studies would help to further understand the molecular mechanisms underlying anti-mitotic agents-induced apoptosis, and provide the rationale for developing specific Cdc20 inhibitors as effective anti-cancer therapies. In Aim #3, we will validate the APCCdc20 E3 ligase as a novel radio-sensitizing target. Taken together, these studies will significantly extend our current knowledge of how depletion of Cdc20 exerts its anti-tumor biological functions through controlling the abundance of Bim. Our proposed studies will provide the molecular basis for designing novel strategies to target Cdc20 by radiation for achieving better clinical treatment of cancer patients.